Human alveolar macrophages play a critical role in the response of the lung to sepsis and the development of acute lung injury. The major stimulus for the activation of these cells in the setting of sepsis is the recognition of microbial products such as endotoxin or lipopolysaccharide (LPS) from gram-negative bacteria and peptidoglycan (PC) from gram-positive bacteria. A number of signaling pathways play a role in the alveolar macrophage response to LPS. We have recently defined MAP kinase pathway roles in LPS signaling, transcription factor activation, and production of cytokines. We found that activation of all three MAP kinase pathways (ERK, JNK, p38) was needed for optimal release of TNF. Some of the results of these studies led us to investigate another important signaling pathway, the phosphatidylinositol 3-kinase (PI 3-kinase) pathway. Although this grant will not lose our interest in MAP kinase signaling, the overall focus of this project is to define the role of the PI 3-kinase pathway in LPS-mediated signaling in human alveolar macrophages. We wish to define the mechanisms for activation of PI 3-kinase (Aim 1), and the downstream effects of PI 3-kinase activity (Aim 2). The focus for Aim 1 (sphingolipids, reactive oxygen species (ROS) and the PI 3 phosphatase (phosphatase and tensin homologue deleted in chromosome ten, PTEN) derives from the information that ceramide is linked to PI 3-kinase activation (our studies), the described links between PI 3-kinase and ROS, and the fact that PTEN phosphatase activity reverses the effects of PI 3-kinase. The focus for Aim 2 (TNF and PGE2) derives from the information that LPS activation results in the coordinated release of first pro- and then anti-inflammatory mediators and we have already demonstrated a role for PI 3-kinase in this process. The clinical correlate of this sequence is the systemic inflammatory response syndrome (SIRS), which is often followed by a period of immunosuppression and, in some patients, acute lung injury. We have chosen to use TNF and PGE2 as molecular representations of SIRS (TNF) and the period of immunosuppression (PGE2). We wish to pursue this line of study because our preliminary data suggests that positive and negative regulation of the PI 3-kinase pathway is critical for the release of TNF and PGE2. Therefore, the overall hypothesis of this project is that PI 3-kinase activity is a molecular switch that regulates the transition from production of pro- (TNF) to anti- (PGE2) inflammatory mediators.